Neosporosis
N. caninum is a protozoan parasite belonging to the phylum Apicomplexa that includes other significant parasitic pathogens such as Toxoplasma gondii which is closely related to it. N. caninum has been described since 1989 as an agent causing abortion and neonatal mortality in cattle (Thilsted and Dubey. 1989. J. Vet. Diagn. Invest. 1, 205-209), though it can infect a wide range of mammal species (Buxton et al. 2002, Trends Parasitol. 18, 546-552).
Bovine neosporosis is considered to be a parasitic disease of worldwide distribution and one of the most common causes of reproduction failure in the various countries where it was studied (Trees et al. 1999. Int. J. Parasitol. 29, 1195-1200; Anderson et al. 2000, Anim. Reprod. Sci. 60-61, 417-431), including Spain (González et al. 1999. Vet. Rec. 144, 145-150; Pereira-Bueno et al. 2003. Vet. Parasitol. 111, 143-152). The most important clinical manifestation of the infection in pregnant females is abortion that generally occurs between the third and ninth month of pregnancy, and most commonly around 5-6 months. Calves affected that are born alive can show neuromuscular problems, with the first clinical signs appearing at 4-5 days post-delivery, though they can be delayed for up to two weeks. However, most commonly, healthy calves are born, though chronically infected (Dubey and Lindsay, 1999. Vet. Parasitol. 67, 1-59). Furthermore, neosporosis can affect dogs, their definitive host, where it causes polymyositis, encephalitis, paralysis and can cause death (Lindsay and Dubey, 1989. J. Parasitol. 75, 163-165; Buxton et al. 2002. Trends Parasitol. 18, 546-552).
Like T. gondii, the life cycle of N. caninum encompasses three stages. Sporozoites, which infect the intermediate host by intake of the oocysts eliminated by the definitive host. On the other hand, tachyzoites, the fast replication form, responsible for the acute stage of the infection, with the role of dissemination through the host tissues. This process ends when the host develops immunity and a chronic phase is then established, with slow multiplication of the parasite, and formation of tissue cysts with bradyzoites inside, that have been observed in both nervous tissue of several species with natural and experimental infections (Dubey et al., 1988, J. Am. Vet. Med. Assoc. 193, 1259-1263; Dubey et al. 1990. J. Am. Vet. Med. Assoc. 197: 1043-1044; Barr et al. 1992. J. Vet Diagn. Invest. 4, 365-367; Kobayashi et al. 2001. J. Parasitol. 87:434-436) and in the skeletal muscle tissue of dog and cow in natural infections (Peters et al. 2001. Int. J. Parasitol. 31, 1144-1148). These bradyzoites are latent in the cysts of tissues until reactivation (Antony and Williamson. 2001. New Zeal. Vet. J. 49, 42-47; Buxton et al. 2002. Trends Parasitol. 18, 546-552). The mechanisms underlying in the turning of the tachyzoite into bradyzoite and vice versa in parasites of the Apicomplexa group, such as T. gondii or N. caninum, are not known today, but it has been suggested that the immune response can be influencing the latency and reactivation of the infection in animals with chronic infection (Lyons, et al., 2002, Trends Parasitol. 18, 198-201).
With regard to the transmission of the disease, the most recent studies suggest the relatively low importance of post-natal transmission and refer to the persistence, throughout life, of the congenital infection (Davison et al. 1999. Int. J. Parasitol. 29, 1683-1689; Hietala and Thurmond. 1999. Int. J. Parasitol. 29. 1669-1676). Congenital transmission plays a major role with percentages ranging between 50% and 95% (Wouda et al. 1998. Theriogenology 49, 1311-1316; Pereira-Bueno et al. 2000. in: Hemphill and Gottstein (Eds.) Int. J. Parasitol. 30, 906-909) and appears to play a highly significant role in the dissemination and maintenance of the disease (Björkman et al. 1996. J. Am. Vet. Med. Assoc. 208, 1441-1444; Paré et al. 1996 Can. J. Vet. Res. 60, 133-139; Anderson et al. 1997. J. Am. Vet. Med. Assoc. 210, 1169-1172; Schares et al. 1998. Vet. Parasitol. 80, 87-98).
N. caninum Antigens
With regard to the comparison of the antigen composition between the tachyzoite and the bradyzoite of N. caninum, very limited information is available to date, as only one study has been performed that identified specific antigens of the tachyzoite or shared by both stages (Fuchs et al. 1998. J. Parasitol. 84, 753-758), which is in contrast to studies performed in T. gondii, where several specific stage antigens are identified and characterised. The N. caninum antigens include two surface proteins, NcSAG1 (Hemphill et al. 1997, Parasitology, 115, 371-380), specific of tachyzoite, whose gene was cloned by Howe et al. (1998. Infect. Immun, 66, 5322-5328) and NcSRS2 (Hemphill et al. 1996, Parasitol. Res. 82, 497-504), expressed jointly in tachyzoites and bradyzoites. Both surface proteins of N. caninum have been recently tested as subunit vaccines in a murine model (Cannas et al. 2003a. Parasitology 126 (Pt. 4), 303-312). Two microneme proteins, NcMIC3 (Sonda et al. 2000. Mol. Biochem. Parasitol 108, 39-51) and NcMIC1 (Keller et al. 2002. Infect. Immun. 70, 3187-3198), expressed in both stages of the parasite have been recently identified. Furthermore, two sequences of Neospora called NcMIC10 and NcMIC11 have been included in the gene bank, that could code two proteins of microneme, as their sequences are highly comparable to sequences coding the proteins of T. gondii TgMIC10 and TgMIC11, respectively. Other genes that have been cloned are NcGRA6 and NcGRA7, which code proteins of dense granules of the tachyzoite of N. caninum, based on which an ELISA was developed for the diagnosis of the disease (Lally et al. 1996. Clin. Diagn. Lab. Immunol. 3, 275-279). In addition to these proteins, others from dense granules of 29 and 67 kDa, called NcNTPase-I (Asai et al. 1998 Exp. Parasitol. 90, 277-285) and NcGRA2 (Ellis et al. 2000. Parasitology 120 (Pt 4), 383-390), respectively, have been identified and characterised. On the other hand, protein NcMIC3, located in the micronemes of intracellular tachyzoites (Naguleswaran et al. 2001. Infect. Immun. 69, 6483-6494), which gene has been cloned, was expressed as recombinant protein to be used for vaccination purposes (Cannas et al. 2003b. J. Parasitol. 89 (pt. 1) 44-50).
Finally, NcSUB1 is the only cloned gene of N. caninum, that expresses an enzyme. NcSUB1 is a serin-protease of 65 kDa (Louie and Conrad. 1999. Mol. Biochem. Parasitol. 103, 211-223; Louie et al. 2002. J. Parasitol. 88, 1113-1119), located in the micronemes of the tachyzoite, which shows a high amino acid identity with the T. gondii protein called TgSUB1.
With regard to specific antigens of the bradyzoite of Neospora, none have been identified to date, due to the difficulty to obtain cysts with bradyzoites both in vitro and in vivo. However, in T. gondii specific bradyzoite antigens have been described, including a surface antigen (SAG4/p18) which is recognized by a monoclonal antibody (T83B1) directed against a protein of 18 kDa (Tomavo et al. 1991. Infect. Immun. 59, 3750-3753). The gene TgSAG4 encoding this protein in T. gondii has been cloned and characterised by Ödberg-Ferragut et al. (1996, Mol. Biochem. Parasitol. 83, 237-244).
The antigen TgSAG4 T. gondii is a membrane protein anchored by phosphatidylinositol glycans (PI-G). The detection of this specific antigen of the slow growth stage, associated with chronic infection by T. gondii, has a significant diagnostic value as recently shown by Cultrera et al. (2002, Mol. Cell. Probes 16, 31-39). These authors have developed an RT-PCR for the detection of infection by T. gondi, based on the detection of mRNA of the gene TgSAG4 in cerebrospinal fluid of AIDS patients, where T. gondii can cause fatal encephalopathy.
Diagnosis and Prevention of Neosporosis Therefore, since vertical transmission of the disease appears to be the best method of establishment of the disease persistently in exploitations and as neosporosis is one of the main causes of abortion and neonatal mortality in cattle, with the attendant economic losses, control must be mainly aimed at reducing the prevalence of the infection in farms, establishing selective measures for culling and replacement to reduce the number of infected animals.
The etiological diagnosis of abortion in cattle is complex and laborious. In cases where an etiological diagnosis is reached, more than 90% correspond to infectious and parasitic agents where N. caninum currently plays a major role. With regard to the diagnosis of infection by N. caninum, it is essential to perform a laboratory diagnosis to confirm the etiology of the abortion, where serological diagnosis techniques play a major role and provide initial information about the significance of the problem. In adults, the laboratory diagnosis is performed by the detection of specific serum antibodies, which is very useful to establish effective measures for the control of infection, as this type of studies provides highly valuable information about the distribution and frequency of infections in farms (herd seroprevalence) and the risk of abortion due to neosporosis in infected herds (intra-herd seroprevalence) (Thurmond and Hietala. 1995. Parasitol. 81, 364-367; Paré et al. 1996. Can. J. Vet. Res. 60, 133-139).
Therefore, improving the diagnosis is extremely important to accurately establish the health condition of the animals. With this regard, a number of studies have been performed with the aim of validating serological techniques currently used, clarifying some controversial issues, such as the selection of the cut-off point based on the age of the animal and the technique used (Alvarez-Garcia et al. 2003. Vet. Res. 34, 341-352).
As several diagnostic techniques are currently used for N. caninum (Ferre et al. 2003. Res. Adv. Microbiol. 3, 157-167), but none of them allow for distinguishing between a recent and a chronic infection, the identification of specific tachyzoite and bradyzoite antigens, respectively, allows the development of diagnostic techniques that provide more information about the future of the farm in terms of abortions and improve the control of the disease. The expression of these antigens as recombinant proteins and their use for the serological diagnosis of neosporosis provide a highly valuable novel tool. With this regard, diagnostic methods, such as ELISA, have been developed, based on several N. caninum proteins produced in different types of heterologous expression systems (Lally et al. 1996. Clin. Diagn. Lab. Immunol. 3, 275-279; Louis et al. 1997. Clin. Diagn. Lab. Immunol. 4(6), 692-699), but to date none based on specific bradyzoite antigens. Furthermore, the development of monoclonal antibodies and monospecific polyclonal sera against stage specific antigens are an alternative for the diagnosis by the development of a competition ELISA, such as that performed against a protein of 65 kDA of N. caninum tachyzoite (Baszler et al. 1996. J. Clin. Microbiol. 34(6), 1423-1428).
On the other hand, the pharmacological control of neosporosis in cattle is currently unfeasible, there is no experience in the pharmacological treatment of the disease in bovines and the data available are not encouraging. However, the high cost of a possible treatment, doilable are not encouraging. however, the high do uninnive for the diagnosis by the development the appearance of possible resistances and residues in meat or milk, limit chemotherapy as a control measure. Therefore, immunoprophylaxis should be added to the measures for managing the herd.
With this regard, investigations performed recently for the preparation of a vaccine against N. caninum have included the assessment of dead vaccines with variable results, finding some protection against vertical transmission in a murine model (Liddell et al., 1999, J. Parasitol. 85: 1072-1075), but not in a bovine model (Andrianarivo et al. 1999, Int. J. Parasitol. 30:985-990). Immunization with live vaccines, based on less virulent isolates, has been also used (Atkinson et al. 1999. Parasitology 118:363-370) or temperature-sensitive mutants (Lindsay et al. 1999. J. Parasitol. 85: 64-67), in vaccination tests in mice with the aim of stimulating a protective immune response against a fatal infection by N. caninum, obtaining encouraging but not definitive results, with the problem of originating persistently infected animals. On the other hand, the vaccines of subunits show a number of advantages over traditional vaccines (Jenkins, 2001. Vet. Parasitol. 101, 291-310), including the safety and relative stability of recombinant proteins, compared to live parasites, the flexibility of including only antigens inducing a protective immune response, and the ability to establish large-scale production. The development of subunit vaccines for the prevention of infection, abortion or vertical transmission of the infection, based on N. caninum antigens, provides a new tool for the control of the same. For the moment, very few studies have been performed on the matter. Only tachyzoite antigens or shared by both stages have been used, such as recombinant proteins expressed and purified from a prokaryote system, including NcMIC3, that induced protective immunity against cerebral neosporosis in a murine model (Cannas et al. 2003. J. Parasitol. 89(1), 44-50), as well as NcSAG1 and NcSRS2, two surface proteins of tachyzoite, inoculated in the same model combined as recombinant antigens and DNA vaccines (Cannas et al. 2003. Parasitology 126, 303-312), obtaining good results. Plasmids expressing NcGRA7 protein or NcsHSP33 protein have been recently used as DNA vaccines in a murine model, obtaining a partial protection against congenital transmission of the infection (Liddell et al. 2003. J. Parasitol. 89(3), 496-500). However, to date the development of these vaccines has not been based on specific antigens of the bradyzoite stage, because the first described in N. caninum is that produced by the gene NcSAG4 as shown in the description of the invention of this report.
As set out in the background, the isolation of genes expressed specifically in each stage is highly relevant for the study of this disease and the molecular mechanisms determining the establishment of the chronic infection, and the reactivation of the infection, improving the understanding and control of the disease. With this regard, the isolation of the NcSAG4 gene in N. caninum, homologous of the TgSAG4 gene, coding a specific protein of the bradyzoite stage in T. gondii, its cloning and the expression of stage specific antigen NcSAG4 as recombinant protein, as set out in the description of the invention hereof, provides a highly valuable solution for the diagnosis of neosporosis, and for the analysis of the pathogenesis of the disease, and its use as a vaccine is an alternative for the control of the same.